Citrus Aurantium L.)

processes

Article Yield, Phytochemical Constituents, and Antibacterial Activity of Essential Oils from the Leaves/Twigs, Branches, Branch Wood, and Branch Bark of Sour Orange (Citrus aurantium L.)

Mohammad K. Okla 1, Saud A. Alamri 1, Mohamed Z.M. Salem 2,* , Hayssam M. Ali 1,3, Said I. Behiry 4, Ramadan A. Nasser 2, Ibrahim A. Alaraidh 1, Salem M. Al-Ghtani 5 and Walid Soufan 6

1 Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; [email protected] (M.K.O.); [email protected] (S.A.A.); [email protected] (H.M.A.); [email protected] (I.A.A.) 2 Forestry and Wood Technology Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21545, Egypt; [email protected] 3 Timber Trees Research Department, Sabahia Horticulture Research Station, Horticulture Research Institute, Agriculture Research Center, Alexandria 21526, Egypt 4 Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Alexandria 21531, Egypt; [email protected] 5 Biology Department, University College of Taymma, University of Tabuk, Taymma, Tabuk P. O. Box 741, Saudi Arabia; [email protected] 6 Plant Production Department, Faculty of Food & Agriculture Sciences, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; [email protected] * Correspondence: [email protected]; Tel.: +20-1012456137

Received: 11 May 2019; Accepted: 27 May 2019; Published: 11 June 2019

Abstract: In the present work, essential oils (EOs) extracted from different parts of sour orange Citrus aurantium (green leaves/twigs, small branches, wooden branches, and branch bark) were studied through gas chromatography coupled with mass spectrometry (GC/MS). Furthermore, the EOs in the amounts of 5, 10, 15, 20, and 25 µL were studied for their antibacterial activity against three pathogenic bacteria, Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora. The main EO compounds in the leaves/twigs were 4-terpineol (22.59%), D-limonene (16.67%), 4-carvomenthenol (12.84%), and linalool (7.82%). In small green branches, they were D-limonene (71.57%), dodecane (4.80%), oleic acid (2.72%), and trans-palmitoleic acid (2.62%), while in branch bark were D-limonene (54.61%), γ-terpinene (6.68%), dodecane (5.73%), and dimethyl anthranilate (3.13%), and in branch wood were D-limonene (38.13%), dimethyl anthranilate (8.13%), (-)-β-fenchol (6.83%), and dodecane (5.31%). At 25 µL, the EO from branches showed the highest activity against A. tumefaciens (IZ value of 17.66 mm), and leaves/twigs EO against D. solani and E. amylovora had an IZ value of 17.33 mm. It could be concluded for the first time that the wood and branch bark of C. aurantium are a source of phytochemicals, with D-limonene being the predominant compound in the EO, with potential antibacterial activities. The compounds identified in all the studied parts might be appropriate for many applications, such as antimicrobial agents, cosmetics, and pharmaceuticals.

Keywords: GC–MS; hydrodistillation; antibacterial activity; clevenger; Citrus aurantium; phytochemical; essential oils

Processes 2019, 7, 363; doi:10.3390/pr7060363 www.mdpi.com/journal/processes Processes 2019, 7, 363 2 of 15

1. Introduction Natural extracts and essential oils (EOs) extracted from aromatic and indigenous plants have a broad spectrum of biological activities such as antibacterial, antifungal, antioxidant, anticancer [1–8]. EOs from Citrus spp., especially from peels, have been studied extensively in many research projects over the past few decades [9–11]. They have exhibited bioactivity potentials against the growth of pathogenic bacteria, fungi, and insects [12,13]. The main chemical compounds identified in the EOs from Citrus were limonene, α-pinene, β-pinene, citral, linalool, myrcene, γ-terpinene, eugenol methyl ether, neral, geranial, neryl acetate, and β-caryophyllene [14–18]. The Citrus plants have many biological and aromatic properties because of the occurrence of EOs, alkaloids, glycosides, flavonoids, tannins, and other compounds in its various parts [19,20]. Citrus aurantium L. (Rutaceae), known as sour or bitter orange, is extensively consumed in Mediterranean countries as marmalade and a flavoring agent [21]. The extracted oils have been recognized as safe for their wide uses as antibacterial, antifungal antioxidant, anti-inflammatory, and anxiolytic effects [22–25], and have analgesic activity [26]. Limonene was determined as the main component of bitter orange peel EO, followed by β-myrcene, linalool, β-pinene, and α-pinene [27]. The major compound in Tunisian neroli EO extracted from C. aurantium blossoms is 25.7% linalool [28]. The (R)-(-)-linalool was 59–64% in Citrus (south and south-central Brazil), whereas the hydrolate (orange water) of C. aurantium has nootkatone (17%), α-terpineol (10%), linalool (10%), and limonene (0.8%) [29]. At maturity, limonene exhibited the highest level, with several minor compounds, including linalool, myrcene, and α-terpineol, in the EOs from bitter orange peel [30]. Limonene (92–95%) with linalool and linalyl acetate (together 0.3–3.2%) were identified in the EOs from living (fruits that are still on the tree) bitter orange peel [31]. Shen et al. 32] showed the anti-inflammatory potential of EO from blossoms of C. aurantium L. var. amara Engl with major constituents of linalool, α-terpineol, (R)-limonene, and linalyl acetate [32]. C. aurantium zest EO is composed of limonene (85.22%), β-myrcene, and α-pinene as the main compounds [13]. EO of sweet orange zest consisted of limonene as the main compound, followed by myrcene, α-farnesene, and γ-terpinene [33,34], whereas the EO of sweet orange zest from Uganda and Rwanda contained limonene, myrcene, α-pinene, and linalool [35]. Using the hydrodistillation method, the linalool and terpenes were found to be the main compounds in Neroli blossom EO, whereas, in water recovered oils, linalool, linalyl acetate, geraniol, α-terpineol, and nerol were the main compounds [36]. In flowers, the oil showed the presence of camphor, thymol, linalool, carvacrol, and borneol as main compounds with significant anti-oxidant effect [37]. The goal of the present work was to identify the aromatic chemical profile and antibacterial activity of the EOs from different parts of C. aurantium that could be suitable for different industrial purposes.

2. Materials and Methods

2.1. Plant Material of C. aurantium Fresh branches of C. aurantium were collected in 2019, from Alexandria, Egypt, during pruning process for the trees. The resultant materials were separated to leaves/twigs, small green branches, branch wood, and branch bark. The wood and bark of branches were separated. All the materials were washed with tape water to remove the dust, then cut to small pieces by using scissors to facilitate the extraction process of essential oils (EOs).

2.2. Extraction of EOs Approximately 100 g from each of leaves/twigs, branches, the wood of branches, and branch bark from C. aurantium were soaked in 2 L ﬂasks with 1500 mL of water and hydrodistillated for 3 h in a Clevenger-type apparatus [38]. The distillates of the EOs were dried over anhydrous Na2SO4, ﬁltrated, and measured with respect to the mass of fresh weight of raw material (Table1). The EOs Processes 2019, 7, 363 3 of 15 from leaves/twigs (Petitgrain), branches (2–4 cm in diameter), the wood of branches, and branch bark were kept dry in sealed Eppendorf tubes and stored at 4 ◦C prior to chemical analyses.

Table 1. Oil yield from diﬀerent parts of Citrus aurantium.

Oil Yield Part Used (mL/100 g Material) Leaves/twigs 3.45 Branches 1.55 Wood of 1.15 branches Branch bark 1.10

2.3. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis The chemical composition of the essential oils was determined using a Trace GC Ultra-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG–5MS (30 m 0.25 mm 0.25 µm film thickness). Initially, the column oven temperature was held at 45 C, × × ◦ then increased by 5 ◦C/min to 250 ◦C and held for 2 min, then increased to 280 ◦C by 10 ◦C/min. The injector and MS transfer line temperatures were kept at 250 ◦C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 2 min and diluted samples of 1 µL were injected automatically using an Autosampler AS1310 coupled with the GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over a range of m/z of 40–600 in full scan mode. The ion source was set at 200 ◦C. Identification of the constituents was performed on the basis of their retention times and by comparing the mass spectra with those found in the library search (NIST and Wiley) [39]. Type threshold values contained in Xcalibur 3.0 data system of GC/MS were used as match factors and to confirm that all mass spectra are appended to the library with measuring the Standard Index (SI) and Reverse Standard Index (RSI), where the value 650 is acceptable to confirm ≥ the compounds [40].

2.4. Antibacterial Activity Antibacterial evaluation of the EOs was assayed against three phytopathogenic bacteria, Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora (Microbiology Laboratory, Agricultural Botany Department, Faculty of Agriculture (Saba Basha), Alexandria University, Egypt). The antibacterial evaluation test of the studied four EOs was performed by measuring the inhibition zones (IZs) in millimeters around the loaded filter papers with different amounts of oils (5, 10, 15, 20, and 25 µL) using disc diffusion method [40,41]. Sterile filter paper discs (Whatman filter paper no. 1) with a diameter of 4 mm loaded with different amounts of the studied EOs were placed on the surface of prepared agar plates. All the plates were incubated in incubator at 30 ◦C for 24 h. Negative control discs were left without any EO. All of the tests were performed in triplicate and the values of the IZs (the clear zones with no bacterial growth around the loaded discs) were reported including the diameter of the disc.

2.5. Statsitcal Analysis Values of the bacteria’s inhibition zones were statistically analyzed with analysis of variance (ANOVA) in completely randomized design with two factors (oil type and oil amount) using a computer program, Statistical Analysis System [42], and compared with those of the control. Means were compared with L.S.D. test at p < 0.05 levels. Processes 2019, 7, 363 4 of 15

3. Results

3.1. Chemical Composition of the EOs Table2 presents the chemical composition of EOs from C. aurantium green leaves/twigs. The main compounds were 4-terpineol (22.59%), D-limonene (16.67%), 4-carvomenthenol (12.84%), linalool (7.82%), methyl methanthranilate (4.41%), cis-4-thujanol (3.72%), γ-terpinene (3.58%), tetraneurin-α-diol (2.61%), 6,9,12,15-docosatetraenoic acid methyl ester (2.48%), and linalyl acetate (2.28%).

Table 2. Chemical composition of essential oils from Citrus aurantium green leaves and twigs.

Relative Molecular Molecular Compound Quantity Weight SI 1 RSI 2 Formula (%) (g/mol)

Myrcene 0.30 C10H16 136 803 833 β-Pinene 1.21 C10H16 136 804 862 d-Limonene 16.67 C10H16 136 934 936 2-Carene epoxide 0.45 C10H16O 152 793 842 Undecane 0.92 C11H24 156 863 920 γ-Terpinene 3.58 C10H16 136 927 938 cis-4-Thujanol 3.72 C10H18O 154 936 947 Octadecyl vinyl ether 0.76 C20H40O 296 760 766 4-Terpineol 22.59 C10H18O 154 961 966 Dodecane 1.59 C12H26 170 883 883 cis-para-2-Menthen-1-ol 0.71 C10H18O 154 847 886 trans,trans-(+)-5-Caranol 0.52 C10H18O 154 772 841 2,6,10-Trimethyltetradecane 0.56 C17H36 240 768 795 4-Carvomenthenol 12.84 C10H18O 154 932 943 Linalool 7.82 C10H18O 154 839 861 5,9-Dimethyl-4,8-decadienal 0.42 C12H20O 180 770 805 Linalyl acetate 2.28 C12H20O2 196 825 888 α-Terpineol 0.96 C10H18O 154 762 790 Vitamin A aldehyde (Retinal) 0.32 C20H28O 284 704 807 Ascaridol 0.97 C10H16O2 168 765 850 4,7-Octadecadienoic acid methyl ester 0.48 C19H30O2 290 691 712 Arachidonic acid methyl ester 0.54 C21H34O2 318 740 777 Thymol 0.90 C10H14O 150 774 864 6,9,12-Octadecatrienoic acid methyl ester 0.53 C19H32O2 292 719 764 2-(7-Heptadecynyloxy) tetrahydro-2H-pyran 0.83 C22H40O2 336 714 749 (Z)-Pseudosolasodine diacetate 0.94 C31H49NO4 499 680 717 Methyl methanthranilate 4.41 C9H11NO2 165 819 929 30,40,7-Trimethylquercetin 0.41 C18H16O7 344 661 690 2-[4-Methyl-6-(2,6,6-trimethylcyclohex- 1-enyl)hexa-1,3,5-trienyl]cyclohex-1-en- 1.38 C23H32O 324 717 761 1-carboxaldehyde Ethyl iso-allocholate 0.61 C26H44O5 436 717 744 Oleic acid 0.87 C18H34O2 282 685 754 6,9,12,15-Docosatetraenoic acid methyl ester 2.48 C23H38O2 346 713 797 Tetraneurin-α-diol 2.61 C15H20O5 280 697 786 1 SI: Standard Index. 2 RSI: Reverse Standard Index.

Table3 shows the chemical composition of EOs from C. aurantium small green branches. The main compounds in small branches were D-limonene (71.57%), dodecane (4.80%), oleic acid (2.72%), trans-palmitoleic acid (2.62%), undecane (2.28%), 1-nonadecanol (2.11%), γ-terpinene (1.97%), 4-terpineol (2.13%), and α-terpineol (1.04%). Processes 2019, 7, 363 5 of 15

Table 3. Chemical composition of essential oil from Citrus aurantium small branches.

Relative Molecular Molecular Compound Quantity Weight SI 1 RSI 2 Formula (%) (g/mol)

α-Pinene 0.52 C10H16 136 873 934 Decane 0.72 C10H22 142 859 937 Myrcene 1.08 C10H16 136 819 836 2-Methyldodecan-1-ol 0.46 C13H28O 200 788 835 d-Limonene 71.57 C10H16 136 940 941 (E)- 2,3-Epoxycarane 0.49 C10H16O 152 759 817 Undecane 2.28 C11H24 156 928 950 γ-Terpinene 1.97 C10H16 136 878 910 Myristyl alcohol 0.57 C14H30O 214 774 777 1-Nonadecanol 2.11 C19H40O 284 766 775 4-Terpineol 2.13 C10H18O 154 897 942 Dodecane 4.80 C12H26 170 919 934 Tetradecane 0.84 C14H30 198 780 788 α-Terpineol 1.04 C10H18O 154 832 880 3,6-Octadecadienoic acid methyl ester 0.49 C19H34O2 294 729 777 Octahydro- 1,2,4-metheno-1H- 0.46 C H O 164 712 778 cyclobuta[cd]pentalene-3,5-diol 10 12 2 cis-Z-α-Bisabolene epoxide 0.96 C15H24O 220 735 759 Oleic acid 2.72 C18H34O2 282 762 781 Arachidonic acid methyl ester 0.82 C21H34O2 318 753 815 (E)-Acrylic acid, 3-(3-methoxycarbonyl-1-cyclohexen-4- 0.66 C12H16O4 224 604 688 yl)-methylester trans-Palmitoleic acid 2.62 C16H30O2 254 760 807 Ethyl iso-allocholate 0.66 C26H44O5 436 743 772 1 SI: Standard Index. 2 RSI: Reverse Standard Index.

The chemical constituents of C. aurantium branch bark is shown in Table4. The main components were D-limonene (54.61%), γ-terpinene (6.68%), dodecane (5.73%), dimethyl anthranilate (3.13%), undecane (3.00%), tetradecyloxirane (2.08%), ethyl iso-allocholate (1.96%), 4-terpineol (1.59%), myrcene (1.53%), and 1,3-diolein (1.52%).

Table 4. Chemical composition of essential oil from Citrus aurantium branch bark.

Relative Molecular Molecular Compound Quantity Weight SI 2 RSI 1 Formula (%) (g/mol)

α-Pinene 1.28 C10H16 136 884 938 Decane 1.27 C10H22 142 817 929 Myrcene 1.53 C10H16 136 812 841 β-Pinene 1.38 C10H16 136 855 899 2,7-Dimethyl-2,6-octadien-1-ol 0.45 C10H18O 154 703 740 1-Decene 0.52 C10H20 140 765 786 1-Tetradecanol 0.66 C14H30O 214 770 776 d-Limonene 54.61 C10H16 136 938 940 (E)- 2,3-Epoxycarane 0.96 C10H16O 152 774 829 Undecane 3.00 C11H24 156 894 930 γ-Terpinene 6.68 C10H16 136 908 945 cis-p-2-Menthen-1-ol 0.41 C10H18O 154 754 822 Hexahydrofarnesol 1.2 C15H32O 228 750 740 Tetradecyloxirane 2.08 C16H32O 240 743 809 Processes 2019, 7, 363 6 of 15

Table 4. Cont.

Relative Molecular Molecular Compound Quantity Weight SI 2 RSI 1 Formula (%) (g/mol)

4-Terpineol 1.59 C10H18O 154 850 920 Dodecane 5.73 C12H26 170 893 923 2,6,10-Trimethyltetradecane 1.17 C17H36 240 754 782 4-Carvomenthenol 1.20 C10H18O 154 782 800 α-Terpineol 1.15 C10H18O 154 825 884 Methyl hexadecadienoate 0.41 C17H30O2 266 716 723 trans-(Z)-α-Bisabolene epoxide 0.61 C15H24O 220 729 801 4,7-Octadecadienoic acid, methyl ester 0.61 C19H30O2 290 707 730 2-[4-Methyl-6-(2,6,6-trimethylcyclohex- 1-enyl)hexa-1,3,5-trienyl]cyclohex-1- 0.48 C23H32O 324 703 714 en-1-carboxaldehyde Oleic acid 1.33 C18H34O2 282 780 804 9-Hexadecenoic acid 1.16 C16H30O2 254 776 810 Dimethyl anthranilate 3.13 C9H11NO2 165 669 893 Methyl hexadecadienoate 0.92 C17H30O2 266 764 801 1,3-Diolein 1.52 C39H72O5 620 753 780 Ethyl iso-allocholate 1.96 C26H44O5 436 744 767 1 SI: Standard Index. 2 RSI: Reverse Standard Index.

Table5 shows the chemical compounds identiﬁed in C. aurantium branch wood. The main compounds in the EO were D-limonene (38.13%), dimethyl anthranilate (8.13%), (-)-β-fenchol (6.83%), dodecane (5.31%), 4-carvomenthenol (4.21%), γ-terpinene (3.62%), cis-4-thujanol (3.49%), thymol (3.30%), valencene (3.30%), linalool (2.94%), 6,7-dihydrogeraniol (2.15%), and undecane (2.13%).

Table 5. Chemical composition of essential oil from Citrus aurantium branch wood.

Relative Molecular Molecular Compound Quantity Weight SI 1 RSI 2 Formula (%) (g/mol)

α-Pinene 1.50 C10H16 136 941 948 Decane 0.65 C10H22 142 880 939 Myrcene 0.96 C10H16 136 837 906 β-Pinene 1.54 C10H16 136 909 939 D-Limonene 38.13 C10H16 136 940 941 p-Cymene 0.72 C10H14 134 805 823 Undecane 2.13 C11H24 156 934 951 γ-Terpinene 3.62 C10H16 136 901 935 4-Terpineol 0.95 C10H18O 154 866 906 1-Dodecanol 0.54 C12H26O 186 769 798 1-Eicosanol 1.69 C20H42O 298 769 776 Linalool 2.94 C10H18O 154 873 898 cis-4-Thujanol 3.49 C10H18O 154 933 945 Dodecane 5.31 C12H26 170 926 939 7-Methyl pentadecane 1.12 C16H34 226 850 885 4-Carvomenthenol 4.21 C10H18O 154 898 907 Capraldehyde 0.93 C10H20O 156 823 885 (-)-β-Fenchol 6.83 C10H18O 154 932 937 6,7-Dihydrogeraniol 2.15 C10H20O 156 886 897 β-Citrylideneethanol 0.45 C12H20O 180 730 742 trans-Carveol 0.83 C10H16O 152 820 864 (Z)-Citral 1.42 C10H16O 152 782 830 Processes 2019, 7, 363 7 of 15

Table 5. Cont.

Relative Molecular Molecular Compound Quantity Weight SI 1 RSI 2 Formula (%) (g/mol)

6-Methyltetraline 0.57 C11H14 146 777 841 Processes 2019, 7, x FOR PEER REVIEW 7 of 16 Dihydro cuminyl alcohol 0.91 C10H16O 152 805 854 Thymol 3.30 C H O 150 904 917 6-Methyltetraline 0.57 10 14C11H14 146 777 841 DihydroFarnesol cuminyl alcohol 1.05 0.91 C15H26 CO10H16O 222152 801805 854 813 NerolidylThymol acetate 0.663.30 C17H28CO10H2 14O 264150 805904 917 825 ValenceneFarnesol 3.301.05 C15HC2415H26O 204222 931801 813 958 DimethylNerolidyl anthranilate acetate 8.13 0.66 C9H11NO C17H228O2 165264 909805 825 940 Valencene 1 SI: Standard Index.3.30 2 RSI: ReverseC15 StandardH24 Index.204 931 958 Dimethyl anthranilate 8.13 C9H11NO2 165 909 940 1 SI: Standard Index. 2 RSI: Reverse Standard Index. The GC–MS chromatograms of the identiﬁed compounds of EOs from the studied diﬀerent parts of C. aurantiumTheare GC–MS shown chromatograms in Figure1 .of the identified compounds of EOs from the studied different parts of C. aurantium are shown in Figure 1.

Figure 1. Cont.

Processes 2019, 7, 363 8 of 15

Processes 2019, 7, x FOR PEER REVIEW 8 of 16

Figure 1. GC–MSFigure 1. GC–MS chromatogram chromatogram showing showing the the chemical chemical analysis analysis of essential of essential oils from oils leaves/twigs from leaves (1), /twigs (1), small branches (2), bark of branches (3), and wood of branches (4). small branches (2), bark of branches (3), and wood of branches (4). 3.2. Antibacterial Activity of the EOs 3.2. Antibacterial Activity of the EOs From the main effects of the extracted oils from different parts of C. aurantium (Figure 2a), oil Fromfrom the leaves/twigs main effects showed of the the extractedhighest activity oils agai fromnst all di thefferent studied parts three ofphytopathogenicC. aurantium bacteria.(Figure 2a), oil from leavesThe/twigs main effects showed of oil the amount highest from activity all the studied against plant all parts the (Figure studied 2b) showed three phytopathogenic that increasing the bacteria. amount of oil (µL) also increased the antibacterial activity, as measured by the inhibition zone (IZ). The main effects of oil amount from all the studied plant parts (Figure2b) showed that increasing the Table 6 presents the antibacterial activity of the studied EOs from different parts of C. aurantium. amount ofThe oil highest (µL) also activity increased against the the growth antibacterial of A. tumefaciens activity, was asobserved measured by theby application the inhibition of EO from zone (IZ). Table6branches presents at 25 the µL antibacterial(IZ value of 17.66 activity mm), followed of the studiedby oil from EOs leaves/twigs from di atff 20erent and 25 parts µL with of C. IZ aurantium. The highestvalue activity of 15.66 against mm. On the the growthother hand, of A.EOs tumefaciens from bark andwas branch observed wood did by not the show application any activity of EO from branches at 25 µL (IZ value of 17.66 mm), followed by oil from leaves/twigs at 20 and 25 µL with IZ value of 15.66 mm. On the other hand, EOs from bark and branch wood did not show any activity against A. tumefaciens. At 25 µL of leaves/twigs EO, the highest activity (17.33 mm) against D. solani was reported, followed by the application of branch EO at 25 µL (16.66 mm) and 20 µL (16.66 mm). For the antibacterial activity of EOs against the growth of E. amylovora at oil amount of 20 and 25 µL from leaves/twigs, the highest IZ value was observed (17.33 mm), followed by branch EO at 25 µL with IZ value of 15.33 mm. Also, the EO from leaves/twigs at 10 and 15 µL showed good activity against E. amylovora with IZ value of 15.00 mm. Processes 2019, 7, x FOR PEER REVIEW 9 of 16 against A. tumefaciens. At 25 µL of leaves/twigs EO, the highest activity (17.33 mm) against D. solani was reported, followed by the application of branch EO at 25 µL (16.66 mm) and 20 µL (16.66 mm). For the antibacterial activity of EOs against the growth of E. amylovora at oil amount of 20 and 25 µL from leaves/twigs, the highest IZ value was observed (17.33 mm), followed by branch EO at 25 µL withProcesses IZ2019 value, 7, 363 of 15.33 mm. Also, the EO from leaves/twigs at 10 and 15 µL showed good activity9 of 15 against E. amylovora with IZ value of 15.00 mm.

(a) Extracted oil; LS Means Wilks lambda=.00016, F(9, 112.1 )=439.47, p=0.0000 16 A. tumefaciens 14 D. solani E. amylovora 12

4 Inhibition zone (mm) zone Inhibition 2

-2 leaves/twigs Branches Bark-branch Wood-branch Extracted oil

(b) Oil amount; LS Means Wilks lambda=.00004, F(15, 127.39)=326.16, p=0.0000 18 A. tumefaciens 16 D. solani 14 E. amylovora 12 10 8 6 4

Inhibition zone (mm) 2 0 -2 -4 0 5 10 15 20 25 Oil amount (µL)

Figure 2. TheThe main main effects eﬀects of oils oils from from different diﬀerent parts of C. aurantium (a) and their amounts ( b) on the growth of A. tumefaciens , D. solani, and E. amylovora.

Processes 2019, 7, 363 10 of 15

Table 6. Antibacterial activity of essential oils from C. aurantium against three phytopathogenic bacteria.

Oil Amount Inhibition Zone Values (mm) Extracted Oil (µL) A. tumefaciens D. solani E. amylovora Leaves/twigs 0 0.00 0.00 0.00 5 0.00 9.33 0.57 12.66 0.57 ± ± 10 10.00 0.00 14.66 0.57 15.00 0.00 ± ± ± 15 11.66 0.57 15.00 0.00 15.00 0.00 ± ± ± 20 15.66 0.57 16.66 0.57 17.33 0.57 ± ± ± 25 15.66 0.57 17.33 0.57 17.33 0.57 ± ± ± Branches 0 0.00 0.00 0.00 5 0.00 11.33 0.57 12.00 0.00 ± ± 10 6.00 0.00 11.33 0.57 12.00 0.00 ± ± ± 15 10.00 0.00 14.33 0.57 12.33 0.57 ± ± ± 20 10.00 0.00 16.66 1.52 14.66 0.57 ± ± ± 25 17.66 0.57 16.66 0.57 15.33 0.57 ± ± ± Branch bark 0 0.00 0.00 0.00 5 0.00 0.00 6.00 0.00 ± 10 0.00 0.00 10.00 0.00 ± 15 0.00 2.00 3.46 10.00 0.00 ± ± 20 0.00 7.66 0.57 11.66 0.57 ± ± 25 0.00 9.66 0.57 12.33 0.57 ± ± Branch wood 0 0.00 0.00 0.00 5 0.00 0.00 6.00 0.00 ± 10 0.00 10.00 0.00 6.33 0.57 ± ± 15 0.00 10.66 0.57 10.00 0.00 ± ± 20 0.00 10.66 0.57 11.33 0.57 ± ± 25 0.00 13.66 0.57 12.00 0.00 ± ± p-value < 0.0001 < 0.0001 < 0.0001

4. Discussion The results of the present work showed the variation in the chemical composition of the EOs from different parts of C. aurantium. Most previous studies have focused on the identification of chemical composition of EOs from the peels, pericarp, blossoms, and leaves, and no core results have been reported from branches, wood, or bark. Additionally, the trials of antimicrobial activities of the EOs were measured against human pathogenic bacteria and plant pathogenic fungi, with no results about the activity against plant bacterial pathogens. 4-terpineol (22.59%) and D-limonene (16.67%) were the most predominate components abundant in green leaves/twigs of C. aurantium, while D-limonene with percentages of 71.57%, 54.61%, and 38.13% was found in small green branches, branch bark, and branch wood, respectively. Results from Wolffenbuttel et al. [29] showed that limonene (39.5–92.7%) and linalool (14.2–24.8%) are the main components of the pericarp and leaves, respectively, of citrus oils obtained by steam distillation, hydrodistillation, or cold press extraction. Linalyl acetate, linalool, α-terpineol, geranyl acetate, geraniol, and geranial as oxygenated monoterpene hydrocarbons were primarily identified in petitgrain oil of C. aurantium var. amara [12], whereas limonene was present only at a concentration of 1.4%. Processes 2019, 7, 363 11 of 15

Terpinen-4-ol, α-pinene, β-pinene, 1,8-cyneol, linalool, and 4-terpineol and their mixture have been shown to have potent antifungal activity [12,43,44]. The most abundant compounds in Tunisian oil were linalool with lower amounts of linalyl acetate and α-terpineol [45]. Algerian C. aurantium leaf EO showed linalool, γ-terpinene, and α-terpineol with percentages of 18.6, 6.9, and 15.1%, respectively, while in peel EO were linalool, cis-linalool oxide, trans-carveol, endo-fenchyl acetate, and carvone with percentages of 12, 8.1, 11.9, 5.5, and 5.8%, respectively [46]. Previously, α-terpineol from Cinnamomum longepaniculatum decreased cell size and irregular cell shape, cell wall, and membrane of E. coli [47]. α-terpinene, terpinen-4-ol, terpinolene, and α-terpineol had strong antibacterial activities against Propionibacterium acnes and Staphylococcus aureus [48]. Linalyl acetate was present in Sicilian petitgrain oil with a lower amount of linalool [49]. Linalyl acetate and linalool were the main components in petitgrain oil from Turkey [50]. EOs of the peels, flowers, and leaves from C. aurantium, collected from northern Greece, exhibited the primary compounds linalool (29.14%), β-pinene (19.08%), trans-β-ocimene (6.06%), and trans-farnesol (5.14%) [51]. The EOs from blossoms of C. aurantium growing in the Darab region in Fars Province, Iran, showed that geraniol, α-terpineol, linalool, and benzene acetaldehyde were the main compounds [52]. Myrcene was found in low percentage of the present work and previously it was reported that myrcene, which found in the EO, is known to possess cytotoxic activity [53,54]. Dl-limonene with 94.81% is the main compound identified in peel EO from C. aurantium with promising larvicide against Anopheles stephensi [9]. Limonene, (E)-nerolidol, α-terpineol, α-terpinyl acetate, and (E,E)-farnesol were the main compounds in the flower EO of C. aurantium with good antibacterial activity against Pseudomonas aeruginosa [10]. α-terpineol and terpinene-4-ol, found in the leaf EO from C. hystrix, were more active against Acinetobacter baumannii, Streptococcus spp., and Haemophilus influenzae than crude oil, while limonene, the most abundant component of C. hystrix oil, had lower antibacterial activity [55]. Zest EO had limonene (85.22%), β-myrcene (4.3%), and α-pinene (1.29%) as the main components, and the EO showed higher antioxidant activity than did limonene alone with a potential for antibacterial activity against Staphylococcus aureus, Salmonella sp., Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli [13]. Among 34 kinds of citrus EOs, four EOs from C. aurantium zest presented good antioxidant activities, as measured by a DPPH assay [16]. Strong fungicidal activity was exhibited by limonene and (E)-nerolidol present in the EO of the flowers of C. aurantium L. var. amara [56]. Considering that limonene is the major compound of the EO of Citrus, this compound has good antioxidant properties [57]. Additionally, other compounds, such as linalool and borneol, have antitumor effects; sabinene and pinene have anti-inflammatory activity; and citral exhibits analgesic functions [58–62]. Although cis-β-terpineol, D-limonene, 4-carvomenthenol, and linalool were the main compounds in petitgrain EO in the present study, the compounds of linalyl acetate, linalool, α-terpineol, and geranyl acetate [12,18,63] were the main compounds in petitgrain EO, which exhibited good antibacterial and antifungal activity,especially against Bacillus subtilis, Aspergillus niger, and Penicillium expansum, whereas the weakest fungicidal effects were observed for Candida krusei [12]. A mixture of terpenoid containing terpinen-4-ol and linalool exhibited high antifungal activity against Trichophyton mentagrophytes, T. rubrum, Microsporum gypseum, A. niger, and A. flavus [43]. Limonene, linalool, citronellal, and citronellol were the main constituents of EO from C. aurantifolia leaves and fruit peels and exhibited promising antibacterial activity against oral pathogenic bacteria Streptococcus mutans and Lactobacillus casei [64]. Leaves EO of C. aurantium grown in Shiraz (south of Iran) showed the presence of limonene, linalool, and trans-β-ocimene as major components and exhibited strong antioxidant activity [65]. EO obtained by cold pressing of C. aurantium fruits with high percentage of limonene (77.90%) and minor percentages of β-pinene (3.40%) and myrcene (1.81%) was inactive against Escherichia coli and Pseudomonas, while moderately active against Stapylococcus aureus [66]. Limonene from linalool-rich essential oil inhibits S. aureus [67]. The variations in the chemical composition of the EOs could be explained by various extraction processes and plant parts used. Furthermore, they are affected by various soils and climatic characteristics Processes 2019, 7, 363 12 of 15 of the regions where the C. aurantium trees grow [36,45,68–71]. For example, the ranges of linalool acetate, linalool, farnesol, nerolidol, and geranyl acetate at 12.2–28.9%, 22.9–54%, 0.2–10.4%, 0.4–21.4%, and 0.97–9.3%, respectively, in C. aurantium blossom EO were observed by seven different methods of oil extraction [71].

5. Conclusions In the present study, variations in essential oils composition from diﬀerent parts of C. aurantium were reported. 4-terpineol, followed by D-limonene, were the main constituents in EO from the leaves/twigs, while D-limonene was the main constituent in small green branches, the branch wood, and the branch bark. EOs from leaves and small branches promised to be potential antibacterial activates against Agrobacterium tumefaciens, Dickeya solani, and Erwinia amylovora. The EOs obtained from diﬀerent parts of C. aurantium displayed bioactive compounds, which have the potential for application as biopreservative agents, antioxidants, antimicrobial compounds, cosmetics, and pharmaceuticals.

Author Contributions: M.K.O., S.A.A., M.Z.M.S., S.I.B., H.M.A. and R.A.N. designed the experiment, conducted laboratory analyses, wrote parts of the manuscript, and interpreted the results; I.A.A., S.M.A.-G., and W.S. contributed reagents and materials; and M.Z.M.S. visualized and revised the article. Funding: This research was funded by Dean of Scientific Research, King Saud University, through the research group project number PRG-1439-63. Acknowledgments: We extend our appreciation to the Dean of Scientific Research, King Saud University, for funding the work through the research group project number PRG-1439-63. The authors also thank the Deanship of Scientific Research and RSSU at King Saud University for their technical support. Conflicts of Interest: The authors declare no conflict of interest.

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